Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:
- Combine 10–20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H20 (if necessary) to make a 10 µl total reaction volume.
- Denature glycoprotein by heating reaction at 100°C for 10 minutes.
- Make a total reaction volume of 20 µl by adding 2 µl 10X GlycoBuffer 2, 2 µl 10% NP40, 2 µl Neuraminidase, H20 and 1–5 µl O-Glycosidase.
- Incubate reaction at 37°C for 1–4 hours.